Members of the TGFbeta superfamily of growth factors are implicated . in a vast range of biological processes, including the inhibition of cell peroliferation in somatic tissues and the specification of cell fate during embryogenesis. We have identify a novel transcription factor in early Xenopus embryos, FAST-1, which interacts with upstream components of the TGFbeta signal transduction pathway to activate specific TGFbeta responsive promoters. In Xenopus, FAST-1 is a critical regulator of the early embryonic gene program. We now plan to examine the role of mammalian FAST-1 homologs in the regulation of the early embryonic transcriptional program. In preliminary experiments we have cloned mouse and human FAST-1 homologs and found that they bind specifically to Smads and to an activin responsive promoter element, as does Xenopus FAST-1. We also find that mouse FAST, like Xenopus FAST-1, is expressed primarily during early embryogenesis. We now plan to examine the role of mammalian FAT-1 in the regulation of the early embryonic gene program in normal and transformed cells. First, we will examine the expression of mammalian FASTs and the specificity of coupling of mouse and human FAST to signal transduction downstream of TGFbeta superfamily ligands. We will then examine how ectopic expression of FAST inc ells which normally lack it alters transcriptional responses to TGFbetas. Specifically, we will test the hypothesis that FAST is an important component of the competence of early embryonic cells to express early mesodermal markers in response to TGFbetas. We will us the ectopic expression of dominant inhibitors of FAST signaling, as well as the knockout of mouse FAST by homology recombination in ES cells, to test whether FAST is necessary for the expression of early embryonic mesodermal gene program in response to TGFbeta stimulation. We will also examine how inhibitor of the TGFbeta/FAST signaling pathway affects the ability of aggregated ES or DMSO-treated EC cells to differentiate into mesoderm. These experiments will elucidate how TGFbetas regulate distinct cellular response in embryonic and post-embryonic tissues, and the role of the expression of embryonic genes in the differentiation of embryonal stem cells.
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